Porous CoFe₂O₄ was prepared via a simple and controllable method to develop a low-cost, high-efficiency, and good-stability nanozyme. The morphology and microstructure of the obtained CoFe₂O₄ was investigated by X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM), high-resolution TEM (HRTEM), specific surface area and pore analysis, and Raman spectroscopy. The results show that the annealing temperature has an important effect on the crystallinity, grain size, and specific surface area of CoFe₂O₄. CoFe₂O₄ obtained at 300 °C (CF300) exhibits the largest surface area (up to 204.1 m² g⁻¹) and the smallest grain size. The peroxidase-like activity of CoFe₂O₄ was further verified based on the oxidation of peroxidase substrate 3,3’,5,5’-tetramethylbenzidine (TMB) in the presence of H₂O₂. The best peroxidase-like activity for CF300 should be ascribed to its largest surface area and smallest grain size. On this basis, an effective method of colorimetric detection H₂O₂ was established. In addition, the porous CoFe₂O₄ was also used for the catalytic oxidation of methylene blue (MB), indicating potential applications in pollutant removal and water treatment.

In this study, we used high-speed countercurrent chromatography (HSCCC) to separate two bioactive polyphenols, rhaponticin and isovitexin, present in fenugreek (Trigonella foenum-graecum L.) seeds. The HSCCC process employed a two-phase solvent system composed of chloroform, methanol, water, and n-butanol in a 4:3:2:1.5 ratio (v:v:v:v). The HSCCC separation conditions were optimized using response surface methodology (RSM). The optimal separation conditions were as follows: separation temperature, 26°C; revolution speed, 874 rpm; flow rate, 1.3 mL/min. Under these conditions, 21.6 mg of rhaponticin and 12.8 mg of isovitexin (>96.0% purity) were obtained from 100 mg of the crude fenugreek seed extract. The purities and structures of the separated compounds were determined by high-performance liquid chromatography (HPLC), and both proton and carbon nuclear magnetic resonance spectroscopy (¹H NMR and ¹³C NMR, respectively). The results demonstrate that (i) HSCCC is an efficient technique for separating polyphenol compounds from seed extracts of fenugreek (Trigonella foenum-graecum L.) and (ii) RSM is an effective approach for optimizing HSCCC separation conditions.